Visceral Leishmaniasis in a Dog: Clinical, Hematological and Pathological Observations L. Tryphonas, Z. Zawidzka, M. A. Bernard and E. A. Janzen*
ABSTRACT Visceral leishmaniasis was diagnosed in a dog that had been living with his owners in Spain for two years. Clinical diagnosis was somewhat delayed as the disease is largely unknown to Canada and was manifested by a nonresponsive anemia which was not easily explained on peripheral blood evaluation alone, and concomitant interstitial nephritis. On post mortem examination splenomegaly was the main gross pathological finding. Light microscopic examination of bone marrow aspirates and subsequent electron microscopic examination of splenic and hepatic tissues revealed numerous Leishman-Donovan bodies in cells of the reticuloendothelial system. Parasitized reticuloendothelial cells were seen singly or forming granulomata. These latter did not contain giant cells and were confined mainly to the liver and spleen, being sparse and single in the first but extremely numerous and coalescing in the latter. Accumulation of intrafollicular hyaline material was seen in a small number of splenic follicles. Leishman-Donovan bodies on electron microscopic examination had a trilaminar periplast, a large round nucleus with heavy blocks of marginated chromatin and two nucleoli, a short flagellum and a kinetoplast. Lymph nodes and bone marrow had numerous parasitized macrophages but no granulomata. Leishman-Donovan bodies were not detected in the lungs and kidneys both of which exhibited a chronic interstitial reaction. The comparative hematological profile as well as the importance of bone marrow and electron microscopic examinations of the spleen and liver in diagnosis are discussed. The potential public health hazard of leishmaniasis to North America and particularly to Canada is considered. *Pathology Section, Research Laboratories, Health Protection Branch, Department of National Health and Welfare, Tunney's Pasture, Ottawa, Ontario KlA OL2 (Tryphonas and Zawidzka) and Alta Vista Animal Hospital, 1814 Bank Street, Ottawa, Ontario K1V 7Y6 (Bernard and Janzen). Submitted November 14, 1975.
Volume 41
-
January, 1977
RESUME Les auteurs ont diagnostique un cas de leishmaniose, chez un chien qui avait passe deux ans en Espagne avec ses maitres. L'etablissement tardif du diagnostic clinique de la condition s'explique par le fait que la leishmaniose n'existe pratiquement pas au Canada et qu'elle se manifeste par une anemie refractaire qu'on s'expliquait difficilement en ne se basant que sur des examens hematologiques; de plus, le chien souffrait simultanement de nephrite interstitielle. Lors de la necropsie, la principale lesion consistait en une splenomegalie. L'examen microscopique d'aspirations de moelle osseuse et des examens ulterieurs du foie et de la rate, 'a I'aide de la microscopie electronique, reveflerent la presence de plusieurs corps de Leishman-Donovan dans les cellules du systieme reticulo-endothelial. Les cellules parasitees se presentaient individuellement ou sous la forme de granulomes. Ceux-ci ne contenaient pas de cellules geantes et se confinaient surtout dans le foie et la rate; rares et individuels dans le foie, ils etaient par ailleurs tres nombreux et fusionnes, dans la rate. La pulpe blanche splenique contenait un peu de substance hyaline. La microscopie electronique rev6la que les corps de Leishman-Donovan possedaient un periplaste trilaminaire, un gros noyau rond, pourvu de points denses peripheriques de chromatine et de deux nucleoles, un flagelle court et un cinetoplaste. Les ganglions lymphatiques et la moelle osseuse contenaient plusieurs macrophages parasites, mais pas de granulomes. Les auteurs ne decel'erent pas de corps de Leishman-Donovan dans les poumons ou les reins; ces organes presentaient cependant une reaction proliferative interstitielle. Ils commentent le profil hematologique comparatif, ainsi que l'importance diagnostique de l'etude de la moelle osseuse et de 1'examen au microscope electronique de la rate et du foie. Ils disent aussi quelques mots sur le danger eventuel de la leishmaniose pour la sante publique en Amerique du Nord, particulierement au Canada.
1
INTRODUCTION Leishmaniasis caused by Leish mania donovani is a geographically restricted, arthropod transmitted protozoan zoonosis affecting among other mammals dogs and man (11, 27, 28, 29). It is a disease of the reticuloendothelial system (RES) and is best known to be enzootic and endemic in parts of the Americas, Near East as well as in peri-Mediterranean countries from where it is occasionally imported into countries known to be free of the parasite (13, 15, 16, 18, 27, 28, 29). Its true distribution however is much more world-wide (9, 25, 31. 33). Although several forms of the disease have been described, visceral or systemic leishmaniasis (Kala-azar) is the severest and commonest form in both man and dog (7, 11, 27). The importance of canine leishmaniasis lies in the well established fact that, in enzootic areas where sandfly vectors (species of Phlebotomus) and Leishmania donovani co-exist, dogs are an important reservoir host from which the Leishmania can easily spill over to the human population. It has been indicated however that in the case of man the disease can be transmitted also by contact (27). Diagnosis of leishmaniasis has been traditionally based on the demonstration of the organism and/or lesions by histopathological, culture, animal inoculation and serological techniques (1, 18), but it is often delayed in disease free areas. Electron microscopic examination is a recent adjunct to the armamentarium of diagnostic methods (4, 6). The purpose of the present report is to describe the clinical, hematological and pathological observations on imported canine leishmaniasis, discuss the significance of bone marrow evaluation and electron microscopic examination of the hemopoietic and other tissues in the diagnosis of cases in which circumstances preclude biological tests and compare existing similarities andi dissimilarities between human and canine leishmaniasis.
with listlessness and the complaint of pain in the left hind leg, and slow but progressive loss of body weight over a period of two months. Past history revealed that the dog had travelled extensively, having had a fracture repaired in Quebec City in 1967, being spayed in Dartmouth, Nova Scotia in 1968 and having been in the village of Mijas, Spain from September 1970 to September 1972. Clinical examination revealed fair bodily condition, dull hair coat, slight pallor of visible mucous membranes and swelling of the left femorotibial joint with crepitation on flexion. The results of fecal examination for parasites were negative. Roentgenograms revealed osteoarthrosis of the left coxofemoral and femorotibial joints. Urine analysis and peripheral blood evaluation results were interpreted to indicate that the dog had normochromic, normocytic anemia and nephropathy (Table I). Hematinic and supportive treatment was prescribed and the dog was released. Subsequently the dog was admitted repeatedly for further examination including exploratory laparotomy and treatment as she was not responding satisfactorily and developed new signs such as cough, bilateral iritis and uveitis, weakness, anorrhexia, vomiting, loss of weight and persistent anemia (Table II). On November 14, blood serum analysis revealed elevated total protein with hypoalbuminemia, hyperglobulinemia and a markedly lowered A-G ratio (Table III). On November 17, leishmaniasis was diagnosed when numerous Leishmania organisms were detected in bone marrow smears (Table IV). Antimonic treatment, instituted promptly, was partially successful. The dog's health improved considerably with some remissions for approximately five months but deteriorated rapidly during the last five days of life. The dog died on April 16, 1973.
MATERIALS AND METHODS HISTORY AND CLINICAL FINDINGS On October 2, 1972 a six year old. spayed. black Labrador Retriever dog was presented
2
Blood samples were taken in 15%' EDTA tripotassium salt as an anticoagulant at a concentration of 0.01 ml/1.0 ml of blood. Erythrocyte (RBC) and total leukocyte (WBC) counts as well as mean corpuscular
Can. J. comp. Med.
TABLE I. Canine Leishmaniasis. Results of Repeated Urine Examinations over a Period of Three Months 2 October 1972
Analysis Specific gravity pH.................... Protein mg/dl ............. Glucose, bilirubin, ketones. . Microscopic Examination
1037 6.5 300 Neg
Dates 7 November 14 November 1972 1972
1030 6.5 100 Neg
1054 7.0 300 Neg
few
few few none seen
few few few granular
occasional
few
Neg
occasional
....................
none seen
occasional granular
some
some
Casts
Epithelial cells ............ ah.p.f. = high power field
1973
1050 6.0 300
RBC/h.p.f.5 ............... WBC/h.p.f ................
none seen
3 January
some
TABLE II. Evaluation of Peripheral Blood During the Course of Canine Leishmaniasis
Erythrocytes x 1012/1
......
Hemoglobin gms/dl ........ Hematocrit 1/1 ............ MCV fl ................... MCH pg ................. MCHC g/dl .............. Reticulocytes % of 3000 cells Total leukocytes x 109/1 ...
Basophils x 109/1 ........ Eosinophils x 109/1 Neutrophils (bands) x i09/1 ...............
Neutrophils (segm) x 109/1 ...............
Lymphocytes x 109/1 Monocytes x 109/1
Number of cells counted Nucleated red cells/200 WBC .................. -Brackets = %7c values
16 November 1972 3.22 8.3
19 December 1972 3.12
0.25 74 26 33 13.300 0 (0.0). 0.067 (0.5)
8.1 0.23 74 26 35 0.8 7.200 0 (0.0) 0.400 (5.5)
0.467 (3.5) 10.100 (76.0) 0.933 (7.0) 1.730 (13.0) 200
0.220 (3.0) 5.100 (71.0) 0.865 (12.0) 0.615 (8.5) 200
nane
volume (MCV) and hemotocrit (HCT) values were obtained using a Coulter electronic counter model F. and MCV/HCT accessory.' Hemoglobin (HGB) was determined by a cyanmethemoglobin method using a Coulter hemoglobinometer. Blood smears, for differential counts and morphological assessment; and bone marrow preparations, obtained under general anesthesia from the femur and/or iliac crest, were both stained with Leishman's. Reticulocytes were supravitally prestained with new methylene blue and then counterstained also with Leishman's. Hematoxylin and eosin (H&E) stained sections and wet 10% formalin fixed tissue 'Coulter Electronics Inc., Hialeah, Florida.
Volume 41 - January, 1977
1 metarubricyte
12 April 1973
2 March 1973
3.27 8.6 0.24 74 26 36
2.08 5.1 .041 67 24 36
5.300 0 (0.0) 0.610 (11.5)
7.000 0 (0.0) 0 (0.0)
0.210 (4.0)
0.175 (2.5) 5.950 (85.0) 0.280 (4.0) 0.595 (8.5) 200
2.790 (52.5) 1.030 (19.5) 0.660 (12.5) 200 none
none
TABLE III. Canine Leishmaniasis. Selected, Blood Serum, Biochemical Parameters (14 November 1972) Parameters
Result
Cholesterol mg/dl...... Calcium mg/dl........ Inorganic phosporus mg/dl.............. Total bilirubin mg/dl. Total protein mg/dl.... Albumin gm/dl........ Globulins gm/dl....... A/G................. Uric acid mg/dl....... Urea nitrogen mg/dl. . . Glucose mg/dl........ LDH (Wacker units) Alkaline phosphatase (King Armstrong Units) SGOT (Karmen Units).
350.00 10.00 6.10 1.80 9.50 2.50 7.00 0.35 2.50 5.00 40.00
>350 12 150
Normal Values 125 - 250 9 - 11.5
2.5- 5.0 0 - 0.6 4.9- 7.9 3 - 4.8 1.3- 3.2 > 1 0- 1 10- 20 55- 99 >260 > 10.4 i 3SD 20 -- 35
3
from liver, spleen, bone marrow (femur), joint capsule and adjacent muscle, lungs, lymph node, and small intestine were available for examination. All tissues were processed by conventional methods and embedded in paraffin. Selected serial sections were stained with H&E, periodic acid Schiff reaction (PAS), Leishman, Giemsa, Jone's reticulin, periodic acid methenamine silver (PAMS), Prussian blue and Gridley
fungus methods (17). Formalin prefixed blocks of spleen and liver were immersed in osmium tetroxide, dehydrated through a graded series of methanol and embedded in araldite, Semithin sections stained with 0.5 Cc toluidine blue in 1% borax and ultrathin sections stained with uranyl acetate and lead citrate were examined with a Siemens Elmiscope 101 electron microscope at 80 KV (21).
TABLE IV. Evaluation of Bone Marrow Aspirates During the Course of Canine Leishmaniasis (
values)
17
Total myeloid series ............ Blasts ......................
Progranulocytes ............. Myelocytes neutrophils eosinophils basophils ........ Metamyelocytes neutrophils. eosinophils basophils Bands . ............ Mature neutrophils . ......... Eosinophils . ......... .......... Basophils . ......
......
Total erythroid series ....... . . Rubriblasts .................
Prorubricytes ............... Rubricytes .................. Metarubricytes ..............
Undifferentiated cells .......... Lymphocytes .................. Plasma cells .................. R-E cells ..................... Tissue basophils ...............
Megakaryocytes
...............
Osteoclasts .................... Number of cells counted ........ Myeloid-erythroid ratio ...... R-E cells containing L-D bodies.
Cellularity
....................
November 1972 58.9 2.0 3.4 4.2 (0.3) 0.8 0.0 6.8 0.5 0.0 26.6 13.7 0.9 0.0 6.8 0.1 0.7 4.4 (0.1) 1.6
0.0 10.9 13.8 9.3 0.0 02 0.1 1000 9.6 All Normoplastic
19
December 1972 64.8 1.8 4.0 2.4 (0.2) 0.6 0.0 7.4 0.2 0.0 25.0 21.0 2.4 0.0
19 8 13 January February April 1973 1973 1973 67.0 55.8 58.0 0.8 1.0 1.0 2.8 3.0 4.2 (0.2)a 1.6 (0.4) 2.6 (0.2) 2.4 (0.2) 1.0 1.0 0.0 0.0 0.2 0.2 4.4 6.2 8.2 1.2 1.0 0.0 0.0 0.0 0.0 27.6 23.0 25.6 25.8 14.2 15.0 2.0 3.4 1.4 0.0 0.0 0.0
22.6 0.4 1.0 12.8 (0.6) 8.4
26.2 0.4 2.2 (0.2) 16.4 (0.8) 7.4
0.0 7.2 3.6 1.6 0.0 0.2 0.0 500
0.0 5.6 0.8 0.4 0.0 0.0 0.0 5CO 2.6
2.9 3_4b I1- Ec
lb
1M-B
32.8
0.4 (0.2) 1.4 17.4 0.8 13.6 (0.2) 0.0 8.6
1.6
1.0 0.0 0.2 0.0
5CO
5
1.7 nore seen ILW B
23.0 0.2 1.8 11.4 (0.4) 9.6 0.8 15.0 1.4 1.8 00 0.0 0.0 CO
2.5 1-2') Normoplastic
aBrackets = % cells in mitosis
bExtensive search was required = infiltrated with blood
*OIWB
RESU LTS
HEMATOLOGICAL FINDINGS
Quantitation of erythrocytes and leukocytes, determination of HCT and HGB levels, and morphological assessment of erythrocytes, leukocytes and platelets revealed normocytic predominantly normochromic anemia, normal total WBC, relative
4
and borderline absolute lymphopenia and moderate relative and absolute monocytosis. Morphologically all cellular elements, including platelets were normal. Examination of bone marrow smears suggested cellular but not hyperplastic sample. The differential count indicated severe-
ly impaired erythropoiesis with normal morphology and maturation sequence of the erythroid series. Plasma cells and reticuloendothelial cells were increased in number.
Can. J. comp. Med.
~ ~ andIV).
? The latter contained numerous round or oval 2-4 ,u.m parasites having deeply staining nucleus and rod-like reddish kinetoplast (Fig. 1). Only parasites scattered free near ruptured cells could be studied in detail. They were identified as Leishman-Donovan (L-D) bodies. The myeloid series was mor' 4 phologically and quantitatively normal while U the myeloid erythroid (ME) ratio was t markedly increased (Table IV). ' S , Despite specific and supportive antiane{ mic treatment peripheral blood erythroid # v ; values remained virtually unchanged until the last relapse prior to death. At this time the nonresponsive anemia had become even more severe. The total WBC count fluctuated between normal and borderline low throughout the duration of the disease. Relative neutrophilia persisted except for the occasion when both relative and absolute numbers fell below normal limits. Eosinopenia developed terminally. Absolute granulocyte values were otherwise within normal range. Relative and slight absolute lymphopenia progressed to a marked degree before death. Absolute numbers of mono-
'
.
J,
d
Fig. 2. Splenic follicle with dense cellularity and intrafollicular hyalinosis. PAS. X100.
cytes, initially somewhat elevated, decreased and remained normal until death (Tables II
m~| } ,A
4*
Subsequent post-treatment bone marrow examinations revealed a drastic reduction in the number of reticuloendothelial cells .,few of which contained L-D bodies, normal percentage of plasma cells and increased erythroid activity (Table IV). Eosinophilic precursors were significantly reduced during the terminal relapse (Table IV).
s
^~
sL
GROSS FINDINGS
t
t
Moderate dehydration and emaciation present. The spleen was thickened, .._meaty and had rounded borders. The overall size of the spleen reached at least twice that of normal. The liver was slightly enlarged and was moderately congested. were
J
Fig. 1. Bone marrow smear. Macrophages with large numbers of L-D bodies. Insert: Kinetoplast and nucleus of L-D bodies are clearly discernible. Leishman stain.
LIGHT MIcROSCoPIc FINDINGS The splenic trabeculae were prominent
.A0OD.
Volume 41
-
January, 1977
5
{,_.
while the white pulp consisted of dense enlarged lymphoid follicles, some with distinct germinal centers and several with intrafollicular hyalinosis (5) (Fig. 2). The red pulp was densely cellular and contained little blood (Fig. 3). The increased cellularity of the red pulp was due to a florid proliferation of macrophages that gave rise to granulomata of single cell type without giant cells (Fig. 5). The granulomata were often surrounded by a variably thick layer of lymphocytes, lymphoblasts, and plasma cells (Fig. 4). Proliferating histiocytes harboured in their cytoplasm numerous L-D bodies. These tended to be mostly round or occasionally C-shaped dark staining bodies infrequently surrounded by a halo (Fig. 5). Parasitized macrophages were ubiquitous except within lymph follicles where they were rarely seen. They accumulated also within the trabecular vessel walls and were prominent subendothelially, from where they were presumably discharged into the lumen (Fig. 6). Some parasitized macrophages harboured hemosiderin pigment, in which the presence of
~~ '~~-~'~~
;ffi
j^bUEs;;b 'S&.S@w6sjv*- i§X{s tA\
Ali.et-,;tti>-W'< Vt
6
3 p
_ 4
.
* 5,
-
Aer I *
Fig. 4. Splenic granuloma (lower right) consisting of a dense accumulation of parasitized macrophages and surrounded by a thin rim of lymphocytes, lymphoblasts and plasma cells. Giant cells are absent. H & E. X100.
iron was confirmed with the Prussian blue reaction as well as with X-ray probe analin a Siemens Elmiscope 101 electron ysis i capsule was thick!microscope. The splenic ened and infiltrated with numerous parasite-laden macrophages. The coarse architecture of the hepatic ~, parenchyma was undisturbed. There was, however, noticeable centrilobular congesB tion and mild periacinar fatty degeneration. E E Close examination revealed enlargement of Kupffer cells and other histiocytes many of which contained numerous L-D bodies and/or hemosiderin, as well as a slight sprinkling of lymphocytes and plasma cells throughout the liver (Figs. 6 and 7a) . Parasitized macrophages tended to form early granulomata. Larger granulomata caused compression atrophy of adjacent hepatic ' a plates (Fig. 7b). Necrosis or proliferation t * of connective tissue and giant cells were absent from such granulomata. It was then concluded that an early granulomatous he-
g^.AB* 2
Fig. 3. Spleen. Thickened capsule and trabeculae with numerous granulomata in the red pulp resulting from of the RES. PTAH. X25.
hyperpslasia
_
patitis
was
developing.
Granulomata were not detected in the lymph nodes, although histiocytes laden
Can. J. comp. Med.
4 j
"'
present in the interstitium (Fig. 8). Evidence of proteinuria characterized by intraluminal accumulation of eosinophilic amorphous material was present in most tubules but not in glomeruli (Fig. 8). L-D bodies were not detected. The small intestine and striated muscle sections were free of parasitized histiocytes in contrast to articular capsule sections in which they were present.
were
ELECTRON MICROScoPE FINDINGS
-C',
; 'A ,- -
Kupffer cells and macrophages of the cords of Billroth as well as monocytes of sthe hepatic and splenic sinusoids contained clusters of L-D bodies. Most LD bodies were free and in intimate contact with the sap of the macrophagic cytoplasm, although some were surrounded to a variable degree by a clear halo (Fig. 9). Only a small number of L-D bodies were seen within menibrane-lined cavities. Irrespective of the type or location of the host cell L-D bodies had similar characteristics (Fig. 10). They were uniform, oval
Fig. 5. Splenic red pulp with numerous heavily parasitized macrophages. PAMS. X925.
with L-D bodies were common in the trabeculae and less so in the sinuses. Although the bone marrow had not fixed well, parasitized macrophages were not difficult to find. No further comments can be made however, on the type of reaction in the marrow except to say that it was cellular. Diffuse interstitial pneumonia was widespread and was characterized by moderate to severe thickening of the alveolar walls, swelling of the alveolar-lining epithelial cells, and accumulation of several macrophages and protein-rich fluid in the alveoli. A search for parasites however was fruitless. The glomeruli had a thickened Bowman's capsule, thickened glomerular tufts, and a modest mesangial cell proliferation. Adhesions of glomerular tufts to Bowman's capsule were not uncommon. The proximal convoluted tubular epithelium had undergone a hydropic change with accumulation of numerous PAS positive granules. The distal and collecting tubular systems were unaffected. Increased fibrosis and prolifercells with few plasma ationation of lymphocytes lasmacells f lymhocyts wlt
Volume 41
-
January, 1977
A
st
t
I
Fig. 6. Splenic vessel. Parasitized cells in endothelial, as well as intraluminal location. H & E. X50.
'ubendothelial and mural
7
were better preserved than others. These included: the Golgi apparatus, a small number of mitochondria, the kinetoplast, a sausage-like structure located near the basal body, lined by two distinct membranes forming cristae mitochondriales-like folds, and containing a dense band of twisted or coiled fibrils embedded in a less dense ground substance (Fig. 10, llc and lld), an amorphous, moderately electron dense material, presumably lipid and a large number of single or aggregated electron dense granules. The nucleus was round with peripheral dense chromatin and two central nucleoli, one with reduced density (Fig. llc). The nuclear membrane was distinct and less electron dense than the chromatin. Parasitized macrophages seemed to be unaffected by the presence of LD bodies and in spite of the prolonged period of formalin fixation appeared reasonably well preserved.
DISCUSSION
Fig. 7. Liver. A. Enlarged Kupffer cells contain numerL-D bodies and/or hemosiderin pigment. B. Slight sprinkling of lymphocytes and plasma cells as well as early granuloma formation by parasitized cells. PAS. X315. ous
structures with mean dimensions of 1.4 x 2.8 ,um in median sections, and had a periplast consisting of a double cytoplasmic membrane lined internally by an array of evenly spaced microtubules (Fig. lla). At the anterior pole of the Ir-D body and diametrically opposed to the nucleus there was a pocket-like invagination of the periplast harbouring the short flagellum. The pocket and flagellum were lined by two membranes only, the tubular array being absent (Figs. llc and lld). The end of the flagellum protruded barely beyond the limits of the anterior pole while its base was continuous with the basal body (Fig. lld). Cross sections of the flagellum revealed nine peripheral doublets and a central pair of fibrils (Fig. llb). The fibrils of the central pair were often found to be less well preserved than those of the nine peripheral doublets. The cytoplasm of the L-D contained a variety of organelles some of which
8
Visceral leishmaniasis was imported into Canada, presumably from Spain, by a dog that had lived in that country for a period of 24 months. Clinical diagnosis was somewhat delayed mainly because the disease is rarely suspected in Canada and was complicated by nephropathy, nonresponsive anemia of unknown etiology, and osteoarthrosis. Thus it proved once more that the iceberg phenomenin, so characteristic of leishmaniasis in its various expressions, is ever present. Laboratory diagnosis of leishmaniasis is rather easy, certain procedures being more demanding than others. Morphological assessment of bone marrow aspirate is of fundamental value especially when examination of peripheral blood smears does not provide a satisfactory explanation of the underlying etiological mechanisms. In our case it proved to be a decisive factor toward accurate diagnosis. Leishmania organisms appear as amastigotes that invade cells of the RES, stain well with Romanowsky type stains, and are easily identifiable by the deeply staining trophonucleus and smaller rod-shaped kinetoplast (30). Histopathological examination at the light and even electron-microscopic levels is fruitful. Spleen, lymph nodes, liver and
Can. J. comp. Med.
__
.
R
in1itochondriales-like folds. The organism can also be recognized by its overall size which ranged slightly around 1.4 x 2.8 ,um (2, 24). Although the number of subpellicular tubular fibrils can be a useful criterion of differentiation between L. donovani and :szL. brasiliensis a precise morphological differentiation from L. tropica is not yet
feasible (2, 10, 14, 23).
..
@
ABSTRACT Visceral leishmaniasis was diagnosed in a dog that had been living with his owners in Spain for two years. Clinical diagnosis was somewhat delayed as the disease is largely unknown to Canada and was manifested by a nonresponsive anemia which was not easily explained on peripheral blood evaluation alone, and concomitant interstitial nephritis. On post mortem examination splenomegaly was the main gross pathological finding. Light microscopic examination of bone marrow aspirates and subsequent electron microscopic examination of splenic and hepatic tissues revealed numerous Leishman-Donovan bodies in cells of the reticuloendothelial system. Parasitized reticuloendothelial cells were seen singly or forming granulomata. These latter did not contain giant cells and were confined mainly to the liver and spleen, being sparse and single in the first but extremely numerous and coalescing in the latter. Accumulation of intrafollicular hyaline material was seen in a small number of splenic follicles. Leishman-Donovan bodies on electron microscopic examination had a trilaminar periplast, a large round nucleus with heavy blocks of marginated chromatin and two nucleoli, a short flagellum and a kinetoplast. Lymph nodes and bone marrow had numerous parasitized macrophages but no granulomata. Leishman-Donovan bodies were not detected in the lungs and kidneys both of which exhibited a chronic interstitial reaction. The comparative hematological profile as well as the importance of bone marrow and electron microscopic examinations of the spleen and liver in diagnosis are discussed. The potential public health hazard of leishmaniasis to North America and particularly to Canada is considered. *Pathology Section, Research Laboratories, Health Protection Branch, Department of National Health and Welfare, Tunney's Pasture, Ottawa, Ontario KlA OL2 (Tryphonas and Zawidzka) and Alta Vista Animal Hospital, 1814 Bank Street, Ottawa, Ontario K1V 7Y6 (Bernard and Janzen). Submitted November 14, 1975.
Volume 41
-
January, 1977
RESUME Les auteurs ont diagnostique un cas de leishmaniose, chez un chien qui avait passe deux ans en Espagne avec ses maitres. L'etablissement tardif du diagnostic clinique de la condition s'explique par le fait que la leishmaniose n'existe pratiquement pas au Canada et qu'elle se manifeste par une anemie refractaire qu'on s'expliquait difficilement en ne se basant que sur des examens hematologiques; de plus, le chien souffrait simultanement de nephrite interstitielle. Lors de la necropsie, la principale lesion consistait en une splenomegalie. L'examen microscopique d'aspirations de moelle osseuse et des examens ulterieurs du foie et de la rate, 'a I'aide de la microscopie electronique, reveflerent la presence de plusieurs corps de Leishman-Donovan dans les cellules du systieme reticulo-endothelial. Les cellules parasitees se presentaient individuellement ou sous la forme de granulomes. Ceux-ci ne contenaient pas de cellules geantes et se confinaient surtout dans le foie et la rate; rares et individuels dans le foie, ils etaient par ailleurs tres nombreux et fusionnes, dans la rate. La pulpe blanche splenique contenait un peu de substance hyaline. La microscopie electronique rev6la que les corps de Leishman-Donovan possedaient un periplaste trilaminaire, un gros noyau rond, pourvu de points denses peripheriques de chromatine et de deux nucleoles, un flagelle court et un cinetoplaste. Les ganglions lymphatiques et la moelle osseuse contenaient plusieurs macrophages parasites, mais pas de granulomes. Les auteurs ne decel'erent pas de corps de Leishman-Donovan dans les poumons ou les reins; ces organes presentaient cependant une reaction proliferative interstitielle. Ils commentent le profil hematologique comparatif, ainsi que l'importance diagnostique de l'etude de la moelle osseuse et de 1'examen au microscope electronique de la rate et du foie. Ils disent aussi quelques mots sur le danger eventuel de la leishmaniose pour la sante publique en Amerique du Nord, particulierement au Canada.
1
INTRODUCTION Leishmaniasis caused by Leish mania donovani is a geographically restricted, arthropod transmitted protozoan zoonosis affecting among other mammals dogs and man (11, 27, 28, 29). It is a disease of the reticuloendothelial system (RES) and is best known to be enzootic and endemic in parts of the Americas, Near East as well as in peri-Mediterranean countries from where it is occasionally imported into countries known to be free of the parasite (13, 15, 16, 18, 27, 28, 29). Its true distribution however is much more world-wide (9, 25, 31. 33). Although several forms of the disease have been described, visceral or systemic leishmaniasis (Kala-azar) is the severest and commonest form in both man and dog (7, 11, 27). The importance of canine leishmaniasis lies in the well established fact that, in enzootic areas where sandfly vectors (species of Phlebotomus) and Leishmania donovani co-exist, dogs are an important reservoir host from which the Leishmania can easily spill over to the human population. It has been indicated however that in the case of man the disease can be transmitted also by contact (27). Diagnosis of leishmaniasis has been traditionally based on the demonstration of the organism and/or lesions by histopathological, culture, animal inoculation and serological techniques (1, 18), but it is often delayed in disease free areas. Electron microscopic examination is a recent adjunct to the armamentarium of diagnostic methods (4, 6). The purpose of the present report is to describe the clinical, hematological and pathological observations on imported canine leishmaniasis, discuss the significance of bone marrow evaluation and electron microscopic examination of the hemopoietic and other tissues in the diagnosis of cases in which circumstances preclude biological tests and compare existing similarities andi dissimilarities between human and canine leishmaniasis.
with listlessness and the complaint of pain in the left hind leg, and slow but progressive loss of body weight over a period of two months. Past history revealed that the dog had travelled extensively, having had a fracture repaired in Quebec City in 1967, being spayed in Dartmouth, Nova Scotia in 1968 and having been in the village of Mijas, Spain from September 1970 to September 1972. Clinical examination revealed fair bodily condition, dull hair coat, slight pallor of visible mucous membranes and swelling of the left femorotibial joint with crepitation on flexion. The results of fecal examination for parasites were negative. Roentgenograms revealed osteoarthrosis of the left coxofemoral and femorotibial joints. Urine analysis and peripheral blood evaluation results were interpreted to indicate that the dog had normochromic, normocytic anemia and nephropathy (Table I). Hematinic and supportive treatment was prescribed and the dog was released. Subsequently the dog was admitted repeatedly for further examination including exploratory laparotomy and treatment as she was not responding satisfactorily and developed new signs such as cough, bilateral iritis and uveitis, weakness, anorrhexia, vomiting, loss of weight and persistent anemia (Table II). On November 14, blood serum analysis revealed elevated total protein with hypoalbuminemia, hyperglobulinemia and a markedly lowered A-G ratio (Table III). On November 17, leishmaniasis was diagnosed when numerous Leishmania organisms were detected in bone marrow smears (Table IV). Antimonic treatment, instituted promptly, was partially successful. The dog's health improved considerably with some remissions for approximately five months but deteriorated rapidly during the last five days of life. The dog died on April 16, 1973.
MATERIALS AND METHODS HISTORY AND CLINICAL FINDINGS On October 2, 1972 a six year old. spayed. black Labrador Retriever dog was presented
2
Blood samples were taken in 15%' EDTA tripotassium salt as an anticoagulant at a concentration of 0.01 ml/1.0 ml of blood. Erythrocyte (RBC) and total leukocyte (WBC) counts as well as mean corpuscular
Can. J. comp. Med.
TABLE I. Canine Leishmaniasis. Results of Repeated Urine Examinations over a Period of Three Months 2 October 1972
Analysis Specific gravity pH.................... Protein mg/dl ............. Glucose, bilirubin, ketones. . Microscopic Examination
1037 6.5 300 Neg
Dates 7 November 14 November 1972 1972
1030 6.5 100 Neg
1054 7.0 300 Neg
few
few few none seen
few few few granular
occasional
few
Neg
occasional
....................
none seen
occasional granular
some
some
Casts
Epithelial cells ............ ah.p.f. = high power field
1973
1050 6.0 300
RBC/h.p.f.5 ............... WBC/h.p.f ................
none seen
3 January
some
TABLE II. Evaluation of Peripheral Blood During the Course of Canine Leishmaniasis
Erythrocytes x 1012/1
......
Hemoglobin gms/dl ........ Hematocrit 1/1 ............ MCV fl ................... MCH pg ................. MCHC g/dl .............. Reticulocytes % of 3000 cells Total leukocytes x 109/1 ...
Basophils x 109/1 ........ Eosinophils x 109/1 Neutrophils (bands) x i09/1 ...............
Neutrophils (segm) x 109/1 ...............
Lymphocytes x 109/1 Monocytes x 109/1
Number of cells counted Nucleated red cells/200 WBC .................. -Brackets = %7c values
16 November 1972 3.22 8.3
19 December 1972 3.12
0.25 74 26 33 13.300 0 (0.0). 0.067 (0.5)
8.1 0.23 74 26 35 0.8 7.200 0 (0.0) 0.400 (5.5)
0.467 (3.5) 10.100 (76.0) 0.933 (7.0) 1.730 (13.0) 200
0.220 (3.0) 5.100 (71.0) 0.865 (12.0) 0.615 (8.5) 200
nane
volume (MCV) and hemotocrit (HCT) values were obtained using a Coulter electronic counter model F. and MCV/HCT accessory.' Hemoglobin (HGB) was determined by a cyanmethemoglobin method using a Coulter hemoglobinometer. Blood smears, for differential counts and morphological assessment; and bone marrow preparations, obtained under general anesthesia from the femur and/or iliac crest, were both stained with Leishman's. Reticulocytes were supravitally prestained with new methylene blue and then counterstained also with Leishman's. Hematoxylin and eosin (H&E) stained sections and wet 10% formalin fixed tissue 'Coulter Electronics Inc., Hialeah, Florida.
Volume 41 - January, 1977
1 metarubricyte
12 April 1973
2 March 1973
3.27 8.6 0.24 74 26 36
2.08 5.1 .041 67 24 36
5.300 0 (0.0) 0.610 (11.5)
7.000 0 (0.0) 0 (0.0)
0.210 (4.0)
0.175 (2.5) 5.950 (85.0) 0.280 (4.0) 0.595 (8.5) 200
2.790 (52.5) 1.030 (19.5) 0.660 (12.5) 200 none
none
TABLE III. Canine Leishmaniasis. Selected, Blood Serum, Biochemical Parameters (14 November 1972) Parameters
Result
Cholesterol mg/dl...... Calcium mg/dl........ Inorganic phosporus mg/dl.............. Total bilirubin mg/dl. Total protein mg/dl.... Albumin gm/dl........ Globulins gm/dl....... A/G................. Uric acid mg/dl....... Urea nitrogen mg/dl. . . Glucose mg/dl........ LDH (Wacker units) Alkaline phosphatase (King Armstrong Units) SGOT (Karmen Units).
350.00 10.00 6.10 1.80 9.50 2.50 7.00 0.35 2.50 5.00 40.00
>350 12 150
Normal Values 125 - 250 9 - 11.5
2.5- 5.0 0 - 0.6 4.9- 7.9 3 - 4.8 1.3- 3.2 > 1 0- 1 10- 20 55- 99 >260 > 10.4 i 3SD 20 -- 35
3
from liver, spleen, bone marrow (femur), joint capsule and adjacent muscle, lungs, lymph node, and small intestine were available for examination. All tissues were processed by conventional methods and embedded in paraffin. Selected serial sections were stained with H&E, periodic acid Schiff reaction (PAS), Leishman, Giemsa, Jone's reticulin, periodic acid methenamine silver (PAMS), Prussian blue and Gridley
fungus methods (17). Formalin prefixed blocks of spleen and liver were immersed in osmium tetroxide, dehydrated through a graded series of methanol and embedded in araldite, Semithin sections stained with 0.5 Cc toluidine blue in 1% borax and ultrathin sections stained with uranyl acetate and lead citrate were examined with a Siemens Elmiscope 101 electron microscope at 80 KV (21).
TABLE IV. Evaluation of Bone Marrow Aspirates During the Course of Canine Leishmaniasis (
values)
17
Total myeloid series ............ Blasts ......................
Progranulocytes ............. Myelocytes neutrophils eosinophils basophils ........ Metamyelocytes neutrophils. eosinophils basophils Bands . ............ Mature neutrophils . ......... Eosinophils . ......... .......... Basophils . ......
......
Total erythroid series ....... . . Rubriblasts .................
Prorubricytes ............... Rubricytes .................. Metarubricytes ..............
Undifferentiated cells .......... Lymphocytes .................. Plasma cells .................. R-E cells ..................... Tissue basophils ...............
Megakaryocytes
...............
Osteoclasts .................... Number of cells counted ........ Myeloid-erythroid ratio ...... R-E cells containing L-D bodies.
Cellularity
....................
November 1972 58.9 2.0 3.4 4.2 (0.3) 0.8 0.0 6.8 0.5 0.0 26.6 13.7 0.9 0.0 6.8 0.1 0.7 4.4 (0.1) 1.6
0.0 10.9 13.8 9.3 0.0 02 0.1 1000 9.6 All Normoplastic
19
December 1972 64.8 1.8 4.0 2.4 (0.2) 0.6 0.0 7.4 0.2 0.0 25.0 21.0 2.4 0.0
19 8 13 January February April 1973 1973 1973 67.0 55.8 58.0 0.8 1.0 1.0 2.8 3.0 4.2 (0.2)a 1.6 (0.4) 2.6 (0.2) 2.4 (0.2) 1.0 1.0 0.0 0.0 0.2 0.2 4.4 6.2 8.2 1.2 1.0 0.0 0.0 0.0 0.0 27.6 23.0 25.6 25.8 14.2 15.0 2.0 3.4 1.4 0.0 0.0 0.0
22.6 0.4 1.0 12.8 (0.6) 8.4
26.2 0.4 2.2 (0.2) 16.4 (0.8) 7.4
0.0 7.2 3.6 1.6 0.0 0.2 0.0 500
0.0 5.6 0.8 0.4 0.0 0.0 0.0 5CO 2.6
2.9 3_4b I1- Ec
lb
1M-B
32.8
0.4 (0.2) 1.4 17.4 0.8 13.6 (0.2) 0.0 8.6
1.6
1.0 0.0 0.2 0.0
5CO
5
1.7 nore seen ILW B
23.0 0.2 1.8 11.4 (0.4) 9.6 0.8 15.0 1.4 1.8 00 0.0 0.0 CO
2.5 1-2') Normoplastic
aBrackets = % cells in mitosis
bExtensive search was required = infiltrated with blood
*OIWB
RESU LTS
HEMATOLOGICAL FINDINGS
Quantitation of erythrocytes and leukocytes, determination of HCT and HGB levels, and morphological assessment of erythrocytes, leukocytes and platelets revealed normocytic predominantly normochromic anemia, normal total WBC, relative
4
and borderline absolute lymphopenia and moderate relative and absolute monocytosis. Morphologically all cellular elements, including platelets were normal. Examination of bone marrow smears suggested cellular but not hyperplastic sample. The differential count indicated severe-
ly impaired erythropoiesis with normal morphology and maturation sequence of the erythroid series. Plasma cells and reticuloendothelial cells were increased in number.
Can. J. comp. Med.
~ ~ andIV).
? The latter contained numerous round or oval 2-4 ,u.m parasites having deeply staining nucleus and rod-like reddish kinetoplast (Fig. 1). Only parasites scattered free near ruptured cells could be studied in detail. They were identified as Leishman-Donovan (L-D) bodies. The myeloid series was mor' 4 phologically and quantitatively normal while U the myeloid erythroid (ME) ratio was t markedly increased (Table IV). ' S , Despite specific and supportive antiane{ mic treatment peripheral blood erythroid # v ; values remained virtually unchanged until the last relapse prior to death. At this time the nonresponsive anemia had become even more severe. The total WBC count fluctuated between normal and borderline low throughout the duration of the disease. Relative neutrophilia persisted except for the occasion when both relative and absolute numbers fell below normal limits. Eosinopenia developed terminally. Absolute granulocyte values were otherwise within normal range. Relative and slight absolute lymphopenia progressed to a marked degree before death. Absolute numbers of mono-
'
.
J,
d
Fig. 2. Splenic follicle with dense cellularity and intrafollicular hyalinosis. PAS. X100.
cytes, initially somewhat elevated, decreased and remained normal until death (Tables II
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Subsequent post-treatment bone marrow examinations revealed a drastic reduction in the number of reticuloendothelial cells .,few of which contained L-D bodies, normal percentage of plasma cells and increased erythroid activity (Table IV). Eosinophilic precursors were significantly reduced during the terminal relapse (Table IV).
s
^~
sL
GROSS FINDINGS
t
t
Moderate dehydration and emaciation present. The spleen was thickened, .._meaty and had rounded borders. The overall size of the spleen reached at least twice that of normal. The liver was slightly enlarged and was moderately congested. were
J
Fig. 1. Bone marrow smear. Macrophages with large numbers of L-D bodies. Insert: Kinetoplast and nucleus of L-D bodies are clearly discernible. Leishman stain.
LIGHT MIcROSCoPIc FINDINGS The splenic trabeculae were prominent
.A0OD.
Volume 41
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January, 1977
5
{,_.
while the white pulp consisted of dense enlarged lymphoid follicles, some with distinct germinal centers and several with intrafollicular hyalinosis (5) (Fig. 2). The red pulp was densely cellular and contained little blood (Fig. 3). The increased cellularity of the red pulp was due to a florid proliferation of macrophages that gave rise to granulomata of single cell type without giant cells (Fig. 5). The granulomata were often surrounded by a variably thick layer of lymphocytes, lymphoblasts, and plasma cells (Fig. 4). Proliferating histiocytes harboured in their cytoplasm numerous L-D bodies. These tended to be mostly round or occasionally C-shaped dark staining bodies infrequently surrounded by a halo (Fig. 5). Parasitized macrophages were ubiquitous except within lymph follicles where they were rarely seen. They accumulated also within the trabecular vessel walls and were prominent subendothelially, from where they were presumably discharged into the lumen (Fig. 6). Some parasitized macrophages harboured hemosiderin pigment, in which the presence of
~~ '~~-~'~~
;ffi
j^bUEs;;b 'S&.S@w6sjv*- i§X{s tA\
Ali.et-,;tti>-W'< Vt
6
3 p
_ 4
.
* 5,
-
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Fig. 4. Splenic granuloma (lower right) consisting of a dense accumulation of parasitized macrophages and surrounded by a thin rim of lymphocytes, lymphoblasts and plasma cells. Giant cells are absent. H & E. X100.
iron was confirmed with the Prussian blue reaction as well as with X-ray probe analin a Siemens Elmiscope 101 electron ysis i capsule was thick!microscope. The splenic ened and infiltrated with numerous parasite-laden macrophages. The coarse architecture of the hepatic ~, parenchyma was undisturbed. There was, however, noticeable centrilobular congesB tion and mild periacinar fatty degeneration. E E Close examination revealed enlargement of Kupffer cells and other histiocytes many of which contained numerous L-D bodies and/or hemosiderin, as well as a slight sprinkling of lymphocytes and plasma cells throughout the liver (Figs. 6 and 7a) . Parasitized macrophages tended to form early granulomata. Larger granulomata caused compression atrophy of adjacent hepatic ' a plates (Fig. 7b). Necrosis or proliferation t * of connective tissue and giant cells were absent from such granulomata. It was then concluded that an early granulomatous he-
g^.AB* 2
Fig. 3. Spleen. Thickened capsule and trabeculae with numerous granulomata in the red pulp resulting from of the RES. PTAH. X25.
hyperpslasia
_
patitis
was
developing.
Granulomata were not detected in the lymph nodes, although histiocytes laden
Can. J. comp. Med.
4 j
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present in the interstitium (Fig. 8). Evidence of proteinuria characterized by intraluminal accumulation of eosinophilic amorphous material was present in most tubules but not in glomeruli (Fig. 8). L-D bodies were not detected. The small intestine and striated muscle sections were free of parasitized histiocytes in contrast to articular capsule sections in which they were present.
were
ELECTRON MICROScoPE FINDINGS
-C',
; 'A ,- -
Kupffer cells and macrophages of the cords of Billroth as well as monocytes of sthe hepatic and splenic sinusoids contained clusters of L-D bodies. Most LD bodies were free and in intimate contact with the sap of the macrophagic cytoplasm, although some were surrounded to a variable degree by a clear halo (Fig. 9). Only a small number of L-D bodies were seen within menibrane-lined cavities. Irrespective of the type or location of the host cell L-D bodies had similar characteristics (Fig. 10). They were uniform, oval
Fig. 5. Splenic red pulp with numerous heavily parasitized macrophages. PAMS. X925.
with L-D bodies were common in the trabeculae and less so in the sinuses. Although the bone marrow had not fixed well, parasitized macrophages were not difficult to find. No further comments can be made however, on the type of reaction in the marrow except to say that it was cellular. Diffuse interstitial pneumonia was widespread and was characterized by moderate to severe thickening of the alveolar walls, swelling of the alveolar-lining epithelial cells, and accumulation of several macrophages and protein-rich fluid in the alveoli. A search for parasites however was fruitless. The glomeruli had a thickened Bowman's capsule, thickened glomerular tufts, and a modest mesangial cell proliferation. Adhesions of glomerular tufts to Bowman's capsule were not uncommon. The proximal convoluted tubular epithelium had undergone a hydropic change with accumulation of numerous PAS positive granules. The distal and collecting tubular systems were unaffected. Increased fibrosis and prolifercells with few plasma ationation of lymphocytes lasmacells f lymhocyts wlt
Volume 41
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January, 1977
A
st
t
I
Fig. 6. Splenic vessel. Parasitized cells in endothelial, as well as intraluminal location. H & E. X50.
'ubendothelial and mural
7
were better preserved than others. These included: the Golgi apparatus, a small number of mitochondria, the kinetoplast, a sausage-like structure located near the basal body, lined by two distinct membranes forming cristae mitochondriales-like folds, and containing a dense band of twisted or coiled fibrils embedded in a less dense ground substance (Fig. 10, llc and lld), an amorphous, moderately electron dense material, presumably lipid and a large number of single or aggregated electron dense granules. The nucleus was round with peripheral dense chromatin and two central nucleoli, one with reduced density (Fig. llc). The nuclear membrane was distinct and less electron dense than the chromatin. Parasitized macrophages seemed to be unaffected by the presence of LD bodies and in spite of the prolonged period of formalin fixation appeared reasonably well preserved.
DISCUSSION
Fig. 7. Liver. A. Enlarged Kupffer cells contain numerL-D bodies and/or hemosiderin pigment. B. Slight sprinkling of lymphocytes and plasma cells as well as early granuloma formation by parasitized cells. PAS. X315. ous
structures with mean dimensions of 1.4 x 2.8 ,um in median sections, and had a periplast consisting of a double cytoplasmic membrane lined internally by an array of evenly spaced microtubules (Fig. lla). At the anterior pole of the Ir-D body and diametrically opposed to the nucleus there was a pocket-like invagination of the periplast harbouring the short flagellum. The pocket and flagellum were lined by two membranes only, the tubular array being absent (Figs. llc and lld). The end of the flagellum protruded barely beyond the limits of the anterior pole while its base was continuous with the basal body (Fig. lld). Cross sections of the flagellum revealed nine peripheral doublets and a central pair of fibrils (Fig. llb). The fibrils of the central pair were often found to be less well preserved than those of the nine peripheral doublets. The cytoplasm of the L-D contained a variety of organelles some of which
8
Visceral leishmaniasis was imported into Canada, presumably from Spain, by a dog that had lived in that country for a period of 24 months. Clinical diagnosis was somewhat delayed mainly because the disease is rarely suspected in Canada and was complicated by nephropathy, nonresponsive anemia of unknown etiology, and osteoarthrosis. Thus it proved once more that the iceberg phenomenin, so characteristic of leishmaniasis in its various expressions, is ever present. Laboratory diagnosis of leishmaniasis is rather easy, certain procedures being more demanding than others. Morphological assessment of bone marrow aspirate is of fundamental value especially when examination of peripheral blood smears does not provide a satisfactory explanation of the underlying etiological mechanisms. In our case it proved to be a decisive factor toward accurate diagnosis. Leishmania organisms appear as amastigotes that invade cells of the RES, stain well with Romanowsky type stains, and are easily identifiable by the deeply staining trophonucleus and smaller rod-shaped kinetoplast (30). Histopathological examination at the light and even electron-microscopic levels is fruitful. Spleen, lymph nodes, liver and
Can. J. comp. Med.
__
.
R
in1itochondriales-like folds. The organism can also be recognized by its overall size which ranged slightly around 1.4 x 2.8 ,um (2, 24). Although the number of subpellicular tubular fibrils can be a useful criterion of differentiation between L. donovani and :szL. brasiliensis a precise morphological differentiation from L. tropica is not yet
feasible (2, 10, 14, 23).
..
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